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European	Journal	of	Chemistry	4	(4)	(2013)	402‐407	

European	Journal	of	Chemistry	
ISSN	2153‐2249	(Print)	/	ISSN	2153‐2257	(Online)		2013	EURJCHEM	

DOI:10.5155/eurjchem.4.4.402‐407.864	

	
	

	

	

European	Journal	of	Chemistry	
Journal	homepage:	www.eurjchem.com	

	 	 	

New	synthetic	benzisoxazole	derivatives	as	antimicrobial,		
antioxidant	and	anti‐inflammatory	agents	

Chalya	Mallappaji	Shivaprasad,	Swamy	Jagadish,	Toreshettahally	Ramesh	Swaroop,																				
Chakrabhavi	Dhananjaya	Mohan,	Rangaswamy	Roopashree,	Kothanahally	Shivaramu	Sharath	Kumar	
and	Kanchugarakoppal	Subbegowda	Rangappa	*	

Department	of	Studies	in	Chemistry,	University	of	Mysore,	Manasagangotri,	Mysore	570	006,	India	

*	Corresponding	author	at:	Department	of	Studies	in	Chemistry,	University	of	Mysore,	Manasagangotri,	Mysore	570	006,	India.	
Tel.:	+91.0821.2419661;	fax:	+91.0821.2500846.	E‐mail	address:	rangappaks@gmail.com	(K.S.	Rangappa).	

	
	

	 	

	 	 	
ARTICLE	INFORMATION	 	 ABSTRACT	
Received:	28	June	2013	
Received	in	revised	form:	19	July	2013	
Accepted:	22	July	2013	
Online:	31	December	2013	

KEYWORDS	

	 A	series	of	piperidine	conjugated	benzisoxazole derivatives	were	synthesized	and	evaluated
for	their	antibacterial,	anti‐oxidant	and	anti‐inflammatory	activities.	The	results	showed	that
most	of	 the	 tested	compounds	exhibit	good	 to	moderate	antimicrobial	activity	against	 some
strains	 of	 Gram	 negative	 bacteria	 (Escherichia	 coli,	Klebsiella	 pneumoniae,	 Salmonella	 typhi,
Shigella	 flexineri)	 and	Gram	positive	bacteria	 (Bacillus	 subtilis).	Further,	 the	molecules	were
evaluated	 for	 anti‐oxidant	 assays	 such	 as	DPPH	 scavenging,	 super	 oxide	 radical	 scavenging
and	hydroxyl	 radical	 scavenging	assays.	Most	 of	 the	 compounds	 showed	potent	 antioxidant
activities.	 Also,	 the	 synthesized	 compounds	 were	 screened	 for	 anti‐inflammatory	 activities
such	 as	 lipoxygenase	 inhibition	 and	 indirect	 haemolytic	 assays,	where	 compounds	 revealed
good	activity.	

Piperidine	
Antioxidant		
Spirolactone	
Antimicrobial		
Benzisoxazole		
Anti‐inflammatory	

	
1.	Introduction	
	

Benzisoxazole	 scaffold	 present	 in	 large	 number	 of	
pharmaceutical	products	with	antimicrobial	[1],	anticonvulsant	
[2,3],	 antitumor	 [4,5],	 antipsychotic	 [6‐8],	 antithrombotic	 [9],	
analgesic	activities	[10].	They	have	also	exhibited	antiglycating	
[11]	 and	 cholinesterase‐inhibiting	 properties	 [12,13].	
Previously	we	have	investigated	various	biological	activities	of	
these	 benzisoxazole	 derivatives	 as	 antimicrobial	 [1]	 and	
cholinesterase‐inhibiting	 agents	 [13].	 In	 continuation	 of	 this	
work,	 we	 report	 herein	 antibacterial,	 antioxidant	 and	 anti‐
inflammatory	 activities	 of	 piperidyl	 spirolactone	 linked	
benzisoxazole	derivatives.	
	
2.	Experimental	
	
2.1.	Instrumentation	

	
The	 melting	 points	 were	 determined	 on	 Selaco	 melting	

point	 apparatus	 and	 are	 uncorrected.	 Infrared	 spectra	 were	
recorded	 on	 Shimadzu	 FT‐IR	 model	 8300	 spectrometer.	 1H	
NMR	 spectra	 were	 recorded	 on	 an	 NMR	 spectrometer	
operating	 at	 400	 MHz	 using	 TMS	 as	 internal	 standard.	 Mass	
spectra	 were	 recorded	 using	 electrospray	 ionization	 mass	
spectrometry.	 The	 C,	 H	 and	N	 analysis	were	 performed	using	
CE‐400	CHN	analyzer.	Reactions	were	monitored	by	TLC	using	
precoated	sheets	of	 silica	 gel	G/UV‐254	of	 0.25	mm	 thickness	
(Merck	 60F254)	 using	 UV	 light	 for	 visualization.	 All	 chemicals	
were	obtained	from	Aldrich,	Fluka	and	Merck	Chemicals.	
	
2.2.	Synthesis	
	

2.2.1.	General	procedure	for	the	synthesis	of	8‐tert‐butyl	4‐
methyl	3‐methyl‐2‐oxo‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐ene‐4,8‐
dicarboxylate	(3)	
	

To	 a	 solution	 of	 tert‐butyl	 4‐oxopiperidine‐1‐carboxylate	
(20	mmol)	 and	 dimethyl	 2‐methylenesuccinate	 (20	mmol)	 in	
THF	 (50	mL),	 a	solution	of	 sodium	methoxide	(40	mmol)	was	
added	 at	 0	 °C.	 The	 reaction	 mixture	 was	 stirred	 at	 room	
temperature	for	8	h.	After	completion	of	the	reaction,	10	mL	of	
water	was	 added;	 the	 organic	 layer	 was	 extracted	with	 ethyl	
acetate	 distilled	 under	 reduced	 pressure	 to	 get	 product	 3	 in	
good	 yield	 (Scheme	 1).	 Colour:	 White.	 Yield:	 70%	 (4.55	 g).	
Viscous	 liquid.	FT‐IR	 (KBr,	 cm‐1):	1555	 (Olefin	C=C	str.),	 1740	
(Ester	 CO	 str.),	 3045	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	
CDCl3,	δ,	ppm):		3.77	(s,	3H,	OMe),	3.30‐3.41	(m,	4H,	CH2),	2.43	
(s,	3H,	CH3),	1.65‐1.80	(m,	4H,	CH2),	1.38	(s,	9H,	(CH3)3).	Anal.	
calcd.	for	C16H23NO6:	C,	59.06;	H,	7.13;	N,	4.31.	Found:	C,	59.15;	
H,	7.21;	N,	4.36%.	MS	(m/z):	326	(M+1).	
		
2.2.2.	General	procedure	for	the	synthesis	of	8‐(tert‐butoxy	
carbonyl)‐3‐methyl‐2‐oxo‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐ene‐4‐
carboxylic	acid	(4)	
	

To	a	solution	of	compound	3	(20	mmol)	in	methanol	30	mL	
and	LiOH	(20	mmol)	 in	water	(30	mL)	was	added	at	0	°C	and	
stirred	 for	 3	 h	 at	 room	 temperature.	 After	 completion	 of	 the	
reaction,	 reaction	 mass	 was	 concentrated	 under	 reduced	
pressure	and	the	residue	was	extracted	with	ethyl	acetate	(3	x	
50	mL),	 the	 solvent	was	 removed	 under	 reduced	 pressure	 to	
get	product	4	 (Scheme	1).	Colour:	White.	Yield:	89%	(5.53	g).	
M.p.:	126‐128	°C.	FT‐IR	(KBr,	 cm‐1):	1715	 (Acid	CO	str.),	1742	
(Ester	CO	str.),	3042	(Aromatic	CH	str.),	3215	(Acid	OH	str.).		



Shivaprasad	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	402‐407	 403	
 

	

 
	

Reagents	and	reaction	conditions:	(a)	MeONa/THF,	0	°C‐RT,	8	h.	(b)	LiOH/MeOH/H2O,	0	°C‐RT,	3	h.	(c)	6‐fluoro‐3‐(piperidin‐4‐yl)benzo[d]isoxazole	
hydrochloride	5,	EDC.HCl/HOBt/DIPEA/CH2Cl2,	0	°C‐RT,	8h.	(d)	HCl/ether,	0	°C‐RT,	1h.	(e)	RCOCl	8,	TEA/EDC,	0	°C‐RT,	3‐4	h.	

	
Scheme	1

	
	
1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	10.5	(s,	1H,	COOH),	3.30‐

3.40	(m,	4H,	CH2),	2.43	(s,	3H,	CH3),	1.65‐1.80	(m,	4H,	CH2),	1.38	
(s,	9H,	(CH3)3).	Anal.	 calcd.	 for	C15H21NO6:	C,	57.87;	H,	6.80;	N,	
4.50.	Found:	C,	57.96;	H,	6.88;	N,	4.58.	MS	(m/z):	311	(M+1).	
	
2.2.3.	General	procedure	for	the	synthesis	of	tert‐butyl	4‐(4‐
(6‐fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐3‐
methyl‐2‐oxo‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐ene‐8‐carboxylate	
(6)	
	

To	 a	 solution	 of	 compound	 4	 (20	 mmol)	 and	 6‐fluoro‐3‐
(piperidin‐4‐yl)benzo[d]isoxazole	 hydrochloride	 [14]	 5	 (20	
mmol)	 in	 dichloromethane	 (40	 mL);	 1‐ethyl‐3‐(3‐dimethyl	
aminopropyl)carbodiimide	hydrochloride	(EDC.HCl)	(20	mmol)	
and	hydroxybenzotriazole	(HOBt)	(2	mmol)	was	added	at	0	°C	
and	the	reaction	mixture	was	stirred	at	room	temperature	for	8	
h.	After	completion	of	the	reaction,	20	mL	of	water	was	added;	
the	organic	layer	was	extracted	with	ethyl	acetate	and	distilled	
under	reduced	pressure	to	get	product	6	in	good	yield	(Scheme	
1).	Colour:	White.	Yield:	78%	(8.00	g).	M.p.:	130‐132	°C.	FT‐IR	
(KBr,	 cm‐1):	 1660	 (Amide	 CO	 str.),	 1742	 (Ester	 CO	 str.),	 3039	
(Aromatic	CH	str.).	1H	NMR	(400	MHz,	CDCl3,	δ,	ppm):	7.54	(d,	
J=7.8	Hz,	1H,	Ar‐H),	7.24	(d,	J=7.8	Hz,	1H,	Ar‐H),	6.97	(s,	1H,	Ar‐
H),	3.30‐3.41	(m,	8H,	CH2),	2.78	(m,	1H,	CH),	2.42	(s,	3H,	CH3),	
1.70‐1.86	 (m,	 8H,	 CH2),	 1.38	 (s,	 9H,	 (CH3)3).	 Anal.	 calcd.	 for	
C27H32FN3O6:	C,	63.15;	H,	6.28;	N,	8.18.	Found:	C,	63.19;	H,	6.35;	
N,	8.26.	MS	(m/z):	513	(M+1).	
	
2.2.4.	General	procedure	for	the	synthesis	of	4‐(4‐(6‐fluoro	
benzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐3‐methyl‐1‐
oxa‐8‐azaspiro[4.5]dec‐3‐en‐2‐one	hydrochloride	(7)	
	

To	a	solution	of	compound	6	(20	mmol)in	diethyl	ether	(40	
mL),	a	saturated	solution	of	HCl	in	ether	was	added	at	0	°C	for	1	
h.	 The	 reaction	 mixture	 was	 concentrated	 under	 reduced	
pressure	 to	get	compound	7	 in	high	yield	 (Scheme	1).	Colour:	
White.	Yield:	92%	(8.26	g).	M.p.:	180‐182	°C.	FT‐IR	(KBr,	cm‐1):	

1664	(Amide	CO	str.),	1744	(Ester	CO	str.),	3035	(Aromatic	CH	
str.),	 3320	 (Amine	 NH	 Str.).	 1H	 NMR	 (400	 MHz,	 DMSO‐d6,	 δ,	
ppm):	7.56	(d,	J=7.8	Hz,	1H,	Ar‐H),	7.24	(d,	J=7.8	Hz,	1H,	Ar‐H),	
6.97	(s,	1H,	Ar‐H),	6.51	(s,	1H,	NH),	3.3‐3.42	(m,	8H,	CH2),	2.78	
(m,	1H,	CH),	2.51	(s,	3H,	CH3),	2.15‐2.30	(m,	4H,	CH2),	1.70‐1.90	
(m,	4H,	CH2).	Anal.	calcd.	for	C22H25ClFN3O4:	C,	58.73;	H,	5.60;	N,	
9.34.	Found:	C,	58.79;	H,	5.68;	N,	9.42.	MS	(m/z):	450	(M+1).	
	
2.2.5.	General	procedure	for	the	synthesis	of	8‐acyl‐4‐(4‐(6‐
fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐3‐
methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐2‐one	(9)	
	

To	a	solution	of	compound	7	 (5	mmol)	and	triethyl	amine	
(5	 mmol)	 in	 dichloromethane	 (20	 mL);	 acyl	 chloride	 (8)	 (5	
mmol)	was	added	at	0	°C	and	stirred	at	room	temperature	for	
3‐4	h.	After	the	completion	of	the	reaction,	20	mL	of	water	was	
added	 and	 extracted	 the	 reaction	 mixture	 with	 dichloro‐
methane	 (2	 x	 20	 mL).	 The	 organic	 layer	 was	 concentrated	
under	reduced	pressure	to	get	products	9	(Table	1)	which	were	
purified	 by	 column	 chromatography	 using	 CHCl3:MeOH	 (9:1,	
v:v)	as	eluent	(Scheme	1).	

8‐(4‐Chlorobenzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)	
piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	 (9a):	 Colour:	White.	 Yield:	 81%	 (2.23	 g).	M.p.:	 108‐110	
°C.	 FT‐IR	 (KBr,	 cm‐1):	 1660	 (Amide	 CO	 str.),	 1740	 (Ester	 CO	
str.),	 3045	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	
ppm):	8.08	(d,	J	=	7.8	Hz,	2H,	Ar‐H),	7.75	(d,	J	=	8.2	Hz,	2H,	Ar‐
H),	7.61	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	7.29	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	
7.03	(s,	1H,	Ar‐H),	3.29‐3.39	(m,	8H,	CH2),	2.72	(m,	1H,	CH),	2.52	
(s,	 3H,	 CH3),	 1.60‐1.90	 (m,	 8H,	 CH2).	 Anal.	 calcd.	 for	
C29H27ClFN3O5:	 C,	 63.10;	 H,	 4.93;	 N,	 7.61.	 Found:	 C,	 63.15;	 H,	
4.96;	N,	7.63%.	MS	(m/z):	553	(M+1).	

8‐(4‐(tert‐Butyl)benzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐
yl)piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐
en‐2‐one	 (9b):	 Colour:	White.	 Yield:	 76%	 (2.17	 g).	 M.p.:	 108‐
110	°C.	FT‐IR	(KBr,	cm‐1):	1671	(Amide	CO	str.),	1749	(Ester	CO	
str.),	 3039	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	



404	 Shivaprasad	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	402‐407	
	
ppm):	7.95	(d,	J	=	7.8	Hz,	2H,	Ar‐H),	7.55	(d,	J	=	7.2	Hz,	1H,	Ar‐
H),	7.47	(d,	J	=	7.8	Hz,	2H,	Ar‐H),	7.24	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	
6.97	(s,	1H,	Ar‐H),	3.28‐3.36	(m,	8H,	CH2),	2.71	(m,	1H,	CH),	2.53	
(s,	 3H,	 CH3),	 1.60‐1.91	 (m,	 8H,	 CH2),	 1.35	 (s,	 9H,	 CMe3).	 Anal.	
calcd.	 for	 C33H36FN3O5:	 C,	 69.09;	 H,	 6.33;	 N,	 7.32.	 Found:	 C,	
69.12;	H,	6.39;	N,	7.36%.	MS	(m/z):	574	(M+1).	

8‐(2,4‐Dichlorobenzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)	
piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	(9c):	Colour:	White.	Yield:	72%	(2.10	g).	M.p.:	114‐116	°C.	
FT‐IR	 (KBr,	 cm‐1):	 1655	 (Amide	CO	str.),	 1756	 (Ester	CO	str.),	
3049	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	 ppm):	
7.75	(s,	1H,	Ar‐H),	7.49‐7.55	(m,	3H,	Ar‐H),	7.26	(d,	 J	=	7.8	Hz,	
1H,	Ar‐H),	7.02	 (s,	1H,	Ar‐H),	3.27‐3.35	 (m,	8H,	CH2),	2.72	 (m,	
1H,	CH),	2.50	 (s,	3H,	CH3),	1.62‐1.88	 (m,	8H,	CH2).	Anal.	 calcd.	
for	C29H26Cl2FN3O5:	C,	59.39;	H,	4.47;	N,	7.17.	Found:	C,	59.45;	H,	
4.52;	N,	7.25%.	MS	(m/z):	587	(M+1).	

8‐(3‐Bromobenzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)	
piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	 (9d):	 Colour:	White.	 Yield:	 83%	 (2.47	 g).	M.p.:	 128‐130	
°C.	 FT‐IR	 (KBr,	 cm‐1):	 1659	 (Amide	 CO	 str.),	 1765	 (Ester	 CO	
str.),	 3055	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	
ppm):	8.25	(s,	1H,	Ar‐H),	8.05	(d,	J	=	7.8	Hz,	1H,	Ar‐H),	7.98	(d,	J	
=	7.6	Hz,	1H,	Ar‐H),	7.55	(m,	2H,	Ar‐H),	7.26	(m,	1H,	Ar‐H),	7.05	
(s,	1H,	Ar‐H),	3.25‐3.35	(m,	8H,	CH2),	2.70	(m,	1H,	CH),	2.52	(s,	
3H,	CH3),	1.60‐1.84	(m,	8H,	CH2).	Anal.	calcd.	for	C29H27BrFN3O5:	
C,	58.40;	H,	4.56;	N,	7.05.	Found:	C,	58.46;	H,	4.60;	N,	7.11.	MS	
(m/z):	597	(M+1).	

8‐(3,5‐Dinitrobenzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)	
piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	 (9e):	 Colour:	White.	 Yield:	 65%	 (1.97	 g).	M.p.:	 184‐186	
°C.	 FT‐IR	 (KBr,	 cm‐1):	 1665	 (Amide	 CO	 str.),	 1771	 (Ester	 CO	
str.),	 3032	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	
ppm):	9.01	(d,	J	=	2.8	Hz,	2H,	Ar‐H),	8.93	(s,	1H,	Ar‐H),	7.59	(d,	J	
=	7.2	Hz,	1H,	Ar‐H),	7.32	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	7.01	(s,	1H,	Ar‐
H),	3.28‐3.39	(m,	8H,	CH2),	2.73	(m,	1H,	CH),	2.49	(s,	3H,	CH3),	
1.62‐1.81	(m,	8H,	CH2).	Anal.	calcd.	for	C29H26FN5O9:	C,	57.33;	H,	
4.31;	N,	11.53.	Found:	C,	57.39;	H,	4.36;	N,	11.59.	MS	(m/z):	608	
(M+1).	

4‐(4‐(6‐Fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐
3‐methyl‐8‐(3,4,5‐trimethoxy	 benzoyl)‐1‐oxa‐8‐azaspiro[4.5]dec‐
3‐en‐2‐one	 (9f):	Colour:	White.	Yield:	70%	(2.12	g).	M.p.:	188‐
190	°C.	FT‐IR	(KBr,	cm‐1):	1660	(Amide	CO	str.),	1765	(Ester	CO	
str.),	 3073	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	
ppm):	7.55	(d,	J	=	7.2	Hz,	1H,	Ar‐H),	7.24	(d,	J	=	7.4	Hz,	1H,	Ar‐
H),	7.17	(s,	2H,	Ar‐H),	6.97	(s,	1H,	Ar‐H),	3.83	(s,	6H,	OMe),	3.81	
(s,	3H,	OMe),	3.25‐3.39	(m,	8H,	CH2),	2.72	(m,	1H,	CH),	2.50	(s,	
3H,	CH3),	1.61‐1.80	(m,	8H,	CH2).	Anal.	calcd.	for	C32H34FN3O8:	C,	
63.25;	 H,	 5.64;	 N,	 6.92.	 Found:	 C,	 63.30;	 H,	 5.71;	 N,	 6.97.	 MS	
(m/z):	608	(M+1).	

4‐(4‐(6‐Fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐
3‐methyl‐8‐(3‐nitrobenzoyl)‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐2‐
one	(9g):	Colour:	White.	Yield:	68%	(1.91	g).	M.p.:	194‐196	°C.	
FT‐IR	 (KBr,	 cm‐1):	 1669	 (Amide	CO	str.),	 1771	 (Ester	CO	str.),	
3061	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	 ppm):	
8.72	(s,	1H,	Ar‐H),	8.51	(d,	J	=	8.0	Hz,	1H,	Ar‐H),	8.42	(d,	J	=	8.0	
Hz,	1H,	Ar‐H),	7.89	(t,	J	=	7.8	Hz,	1H,	Ar‐H),	7.55	(d,	J	=	7.2	Hz,	
1H,	Ar‐H),	7.24	(d,	J	=	7.3	Hz,	1H,	Ar‐H),	6.97	(s,	1H,	Ar‐H),	3.29‐
3.42	(m,	8H,	CH2),	2.68	(m,	1H,	CH),	2.48	(s,	3H,	CH3),	1.65‐1.82	
(m,	8H,	CH2).	Anal.	calcd.	 for	C29H27FN4O7:	C,	61.92;	H,	4.84;	N,	
9.96.	Found:	C,	61.98;	H,	4.89;	N,	9.98.	MS	(m/z):	563	(M+1).	

4‐(4‐(6‐Fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐
8‐(4‐methoxybenzoyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	 (9h):	 Colour:	White.	 Yield:	 72%	 (1.96	 g).	M.p.:	 108‐110	
°C.	 FT‐IR	 (KBr,	 cm‐1):	 1659	 (Amide	 CO	 str.),	 1782	 (Ester	 CO	
str.),	 3047	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	
ppm):	7.92	(d,	J	=	8.0	Hz,	2H,	Ar‐H),	7.55	(d,	J	=	7.6	Hz,	1H,	Ar‐
H),	 7.26	 (d,	 J=7.6	 Hz,	 1H,	 Ar‐H),	 7.16	 (d,	 J=8.0	Hz,	 2H,	 Ar‐H),	
6.97	 (s,	 1H,	 Ar‐H),	 3.82	 (s,	 3H,	 OMe),	 3.32‐3.45	 (m,	 8H,	 CH2),	
2.70	(m,	1H,	CH),	2.49	(s,	3H,	CH3),	1.62‐1.85	(m,	8H,	CH2).	Anal.	
calcd.	 for	 C30H30FN3O6:	 C,	 65.80;	 H,	 5.52;	 N,	 7.67.	 Found:	 C,	
65.85;	H,	5.59;	N,	7.73.	MS	(m/z):	548	(M+1).	

8‐(2,6‐Difluorobenzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)	
piperidine‐1‐carbonyl)‐3‐methyl	 ‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	(9i):	Colour:	White.	Yield:	60%	(1.65	g).	M.p.:	128‐130	°C.	
FT‐IR	 (KBr,	 cm‐1):	 1667	 (Amide	CO	str.),	 1774	 (Ester	CO	str.),	
3059	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	 ppm):	
7.53‐7.56	 (m,	 2H,	 Ar‐H),	 7.19‐7.24	 (m,	 3H,	 Ar‐H),	 6.97	 (s,	 1H,	
Ar‐H),	 3.30‐3.44	 (m,	 8H,	 CH2),	 2.71	 (m,	 1H,	 CH),	 2.54	 (s,	 3H,	
CH3),	 1.60‐1.85	 (m,	 8H,	 CH2).	 Anal.	 calcd.	 for	 C29H26F3N3O5:	 C,	
62.93;	 H,	 4.73;	 N,	 7.59.	 Found:	 C,	 62.96;	 H,	 4.79;	 N,	 7.64.	 MS	
(m/z):	554	(M+1).	

8‐(3‐Chlorobenzoyl)‐4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)	
piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐
2‐one	(9j):	Colour:	White.	Yield:	73%	(2.01	g).	M.p.:	110‐112	°C.	
FT‐IR	 (KBr,	 cm‐1):	 1665	 (Amide	CO	str.),	 1755	 (Ester	CO	str.),	
3068	 (Aromatic	 CH	 str.).	 1H	 NMR	 (400	 MHz,	 CDCl3,	 δ,	 ppm):	
8.22	(s,	1H,	Ar‐H),	8.03	(d,	J	=	7.8	Hz,	1H,	Ar‐H),	7.96	(d,	J	=	7.6	
Hz,	 1H,	Ar‐H),	 7.52	 (m,	2H,	Ar‐H),	7.23	 (m,	1H,	Ar‐H),	7.02	 (s,	
1H,	Ar‐H),	3.26‐3.36	(m,	8H,	CH2),	2.71	(m,	1H,	CH),	2.53	(s,	3H,	
CH3),	1.61‐1.83	(m,	8H,	CH2).	Anal.	 calcd.	 for	C29H27ClFN3O5:	C,	
63.10;	 H,	 4.93;	 N,	 7.61.	 Found:	 C,	 63.10;	 H,	 4.93;	 N,	 7.61.	 MS	
(m/z):	553	(M+1).	
	
Table	1.	Derivatives	of	benzisoxazole.	
Entry R	(8,9) Compound	9	 Yield,	%
1 4‐ClC6H4 9a	 81
2	 4‐CMe3C6H4	 9b	 76	
3	 2,4‐Cl2C6H3	 9c	 72	
4 3‐BrC6H4 9d	 83
5 3,5‐(NO2)2C6H3 9e	 65
6 3,4,5‐(MeO)3C6H2 9f	 70
7 3‐NO2C6H4 9g	 68
8 4‐MeOC6H4 9h	 72
9 2,6‐F2C6H3 9i	 60
10 3‐ClC6H4 9j	 73
	
	
2.3.	Biological	evaluation‐antibacterial,	antioxidant	and	
anti‐inflammatory	activities	
	
2.3.1.	Antibacterial	activity	
	

Antibacterial	 tests	 were	 carried	 out	 by	 disc	 diffusion	
method	using	100	μL	of	suspension	containing	106	cells/mL	of	
bacteria.	The	discs	(6	mm	diameter)	were	impregnated	with	5	
mg	 and	 10	 mg/mL	 of	 each	 compound	 and	 placed	 on	 the	
inoculated	 nutrient	 agar.	 Then,	 the	 inoculated	 plates	 were	
incubated	 at	 37±0.1	 °C	 at	 24	 h.	 One	 antibacterial	 drug,	
chloramphenicol	 was	 used	 as	 positive	 control.	 Antibacterial	
activity	 was	 evaluated	 by	 measuring	 the	 zone	 of	 inhibition	
against	 the	 test	 organisms	 and	 the	 results	 are	 summarised	 in	
Table	2.	
	
2.3.2.	Antioxidant	activity	
	
2.3.2.1.	DPPH	radical	scavenging	assay	
	

DPPH	 radical	 scavenging	 assays	 [15]	 were	 performed	 in	
300	µL	reaction	mixtures	containing	200	µL	of	0.1	mM	DPPH‐
ethanol	 solution,	 90	 µL	 of	 50	mM	 Tris‐HCl	 buffer	 (pH	 =	 7.4),	
and	 10	 µL	 of	 deionised	 water	 (as	 control)	 and	 various	
concentrations	of	 compounds	9a‐j	 (1.8‐9.0	µM).	Ascorbic	 acid	
was	 used	 as	 a	 standard.	 After	 30	 min	 of	 incubation	 at	 room	
temperature,	 absorbance	 (540	 nm)	 of	 the	 reaction	 mixtures	
was	 taken	 by	 a	 plate	 reader	 (Lab	 systems	Mullikan	MS).	 The	
percentage	radical	scavenging	activity	was	calculated	according	
to	Equation	(1).	
	
Inhibition	(%)	=	(Absorbance	control‐Absorbance			
																					Sample/Absorbance	Control)	×	100	 	 (1)	
	

The	 DPPH	 radical	 scavenging	 activity	 is	 demonstrated	 in	
Figure	1	and	Table	3.	
	



Shivaprasad	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	402‐407	 405	
 

	
Table	2.	Antibacterial	activity	of	benzisoxazoles	9a‐j.	
Compound		 Zone	of	inhibition	in	millimetre	*	

Escherichia	coli	 Bacillus	subtilis Klebsiella	pneumoniae Salmonella	typhi	 Shigella	flexneri
9a	 12	 11	 11 10 11	
9b	 13	 12	 9 12 9	
9c	 14	 ‐	 10 ‐ ‐	
9d	 10	 13	 9 12 10	
9e	 16	 15	 16 22 26	
9f	 10	 15	 11 11 9	
9g	 11	 12	 10	 9	 10	
9h	 12	 ‐	 ‐	 ‐	 ‐	
9i	 ‐	 ‐	 10	 ‐	 ‐	
9j	 17	 ‐	 ‐ 11 11	
*	Inhibition	zones	including	disc	(6	mm)	diameter,	Positive	control	zone	is	35	to	40	mm,	“‐“	=	Not	active.	
	
Table	3.	Antioxidant	activity	of	benzisoxazoles	9a‐j.	
Compound	 IC50	values	in	µM	

DPPH	scavenging	assay	 Hydroxy	radical	scavenging	assay	 Superoxide	radical	scavenging	assay	
9a	 7.0	 7.5 7.3
9b	 4.1	 3.9 3.8
9c	 6.2	 6.4 6.5
9d	 7.9	 8.2 8.2
9e	 6.5	 6.2	 6.2	
9f	 4.9	 4.6	 4.1	
9g	 6.8	 6.8 6.8
9h	 5.2	 5.1	 4.7	
9i	 8.5	 8.5	 8.5	
9j	 7.4	 8.0 7.8
Ascorbic	acid	 3.5	 3.4 ‐
Quercetin	 ‐	 ‐ 2.8
“‐“	=	Not	active.	
	
	

	
Figure	1.	DPPH	radical	scavenging	assay.	

	
2.3.2.2.	Hydroxyl	radical	scavenging	assay		
	

The	reaction	mixture	in	final	volume	of	2	mL	containing	0.1	
mL	of	EDTA	(1	mM),	0.01	mL	of	FeCl3	(10	mM),	0.1	mL	of	H2O2	
(10	 mM),	 0.36	 mL	 of	 deoxyribose	 (10	 mM),	 1	 mL	 of	 the	
compounds	9a‐j	 (concentrations	 from	1.8‐9.0	µM),	0.33	mL	of	
phosphate	buffer	(50	mM,	pH	=	7.4)	and	0.1	mL	ascorbic	acid	(1	
mM)	 added	 in	 sequence.	 The	mixture	was	 incubated	 at	 37	 °C	
for	1	h.	1	mL	of	the	incubated	mixture	was	mixed	with	1	mL	of	
10%	 trichloro	 acetic	 acid	 and	 1	 mL	 of	 TBA	 (1%	 in	 0.025	 M	
NaOH),	the	resulting	mixture	was	incubated	in	water	bath	at	90	
°C	 for	 20	 min	 to	 develop	 a	 pink	 chromogen	 which	 was	
measured	at	532	nm	[16].	Ascorbic	acid	was	used	as	a	positive	
control.	Percentage	inhibition	was	evaluated	by	using	Equation	
(2).	
	
Inhibition	(%)	=	(Absorbance	control‐Absorbance					
																			Sample/Absorbance	Control)	×	100	 	 (2)	
	

The	 potency	 of	 benzisoxazoles	 for	 hydroxyl	 radical	
scavenging	activity	is	illustrated	in	Figure	2	and	Table	3.	
	
2.3.2.3.	Superoxide	anion	radical	scavenging	assay	
	

1	mL	of	NBT	(156	µM	NBT	in	100	mM	phosphate	buffer	of	
pH	=	7.4),	1	mL	of	NADH	(468	µM	in	100	mM	phosphate	buffer	
of	pH	=	7.4)	and	varying	concentration	of	compounds	9a‐j	(1.8‐
9.0	µM)	were	mixed	to	give	a	final	volume	of	3	mL.	The	reaction	
was	started	by	the	addition	of	100	µL	of	PMS	(60	µM	in	100	mM	

phosphate	 buffer	 of	 pH	 =	 7.4).	 The	 reaction	 mixture	 was	
incubated	at	25	°C	for	5	min	and	the	absorbance	was	measured	
at	560	nm.	Quercetin	was	used	as	 a	 standard	 [17].	Decreased	
absorbance	 of	 the	 reaction	 mixture	 indicated	 increased	
superoxide	 anion	 scavenging	 activity	 and	 it	 is	 illustrated	 in	
Figure	3	and	Table	3.	
	

	
Figure	2.	Hydroxyl	radical	scavenging	assay.	

	

Figure	3.	Super	oxide	radical	scavenging	assay.	
	
2.3.3.	Anti‐inflammatory	activity	
	
2.3.3.1.	Lipoxygenase	inhibition	assay	
	

Lipoxygenase	 inhibition	 assay	 [18]	 was	 carried	 out	 using	
linoleic	acid	as	substrate	and	lipoxgenase	enzyme.	To	a	solution	
of	0.1	mL	of	0.2	M	borate	buffer	(pH	=	9.0),	containing	0.1	mL	of	
1000	 units	 lipoxidase	 enzyme,	 solution	 of	 compounds	9a‐j	 in	
DMSO	 (1	mg/mL)	was	 added	 and	 incubated	with	 the	 enzyme	
with	various	concentrations	(1.8‐9.0	µM).		



406	 Shivaprasad	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	402‐407	
	

	
Table	4.	Anti‐inflammatory	activity	of	benzisoxazoles	9a‐j.	
Compound	 IC50	values	in	µM	

Lipoxygenase	inhibition	assay PLA2 inhibition	assay	
9a	 8.6	 79.0
9b	 4.1	 37.4
9c	 6.2	 62.7
9d	 7.9	 76.9
9e	 3.9	 40.3
9f	 5.0	 49.8
9g	 3.9	 40.5
9h	 5.2	 53.0	
9i	 8.5	 81.1	
9j	 7.4	 75.3	
Indomethacin	 3.0	 ‐	
Aristolochic	acid	 ‐	 30.0
“‐“	=	Not	determined.	

	
	
The	 tubes	 were	 agitated	 and	 incubated	 at	 room	

temperature	for	5	min,	after	which	2.0	mL	of	substrate	solution,	
0.6	 mM	 linoleum	 acid	 were	 added,	 mixed	 well	 and	 the	
absorbance	was	measured	spectrophotometrically	for	4	min	at	
234	 nm	 (Shimadzu‐2401	 PC).	 Indomethacin	 was	 used	 as	 a	
reference	 standard	 drug.	 Percentage	 (%)	 inhibition	 was	
calculated	by	Equation	(3).		
	
Inhibition	(%)	=	(Absorbance	control‐Absorbance		
																			Sample/Absorbance	Control)	×	100	 	 (3)	
	

Lipoxygenase	 inhibition	 activity	 of	 benzisoxazoles	 is	
summarised	in	Figure	4	and	Table	4.	
	

	
Figure	4.	Lipoxygenase	inhibition	assay.	

	
2.3.3.2.	Inhibition	of	PLA2	induced	haemolysis	in	human	
erythrocytes	
	

The	substrate	for	indirect	hemolytic	activity	was	prepared	
by	suspending	1	mL	of	fresh	human	red	blood	cells	and	1	mL	of	
fresh	Hen’s	egg	yolk	in	8	mL	of	phosphate	buffered	saline.	1	mL	
of	suspension	was	incubated	with	4‐28	μg	of	partially	purified	
venom	for	45	min	at	37	°C	and	the	reaction	was	stopped	by	the	
addition	 of	 9	 mL	 of	 ice	 cold	 PBS.	 The	 suspension	 was	
centrifuged	 at	 2000	 rpm	 for	 20	 min	 and	 then	 the	 released	
haemoglobin	 was	 read	 at	 540	 nm.	 10	 μg	 of	 venom	 sample	
(secretory‐PLA2	 purchased	 from	 sigma)	 was	 incubated	 with	
various	concentration	of	compounds	9a‐j	 (1	mg/mL	in	DMSO)	
for	 30	 min	 at	 room	 temperature	 and	 1	 mL	 of	 substrate	 was	
added,	again	incubated	for	30	min	at	room	temperature	and	the	
reaction	was	stopped	by	adding	9	mL	of	ice	cold	PBS	to	all	test	
tubes	 and	 centrifuged	 at	 2000	 rpm	 for	 10	 min.	 Finally	
absorbance	 was	 measured	 at	 540	 nm	 [19]	 and	 inhibitory	
activities	are	summarised	in	Figure	5	and	Table	4.	
	
3.	Results	and	discussion		

	
Sodium	methoxide	induced	cyclocondensation	of	tert‐butyl	

4‐oxopiperidine‐1‐carboxylate	 (1)	 and	 dimethyl	 2‐methylene	
succinate	 (2)	 in	THF	 to	afford	8‐tert‐butyl	4‐methyl	3‐methyl‐
2‐oxo‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐ene‐4,8‐dicarboxylate	 (3)	 in	
70%	 yield	 (via	 formation	 of	 β‐hydroxy	 ester	 which	 subse‐
quently	 undergo	 intramolecular	 cyclication	 to	 form	 lactone	

with	 exocyclic	 double	 bond.	 Later,	 base	 induced	migration	 of	
double	 bond	 in	 to	 the	 ring	 gives	 compound	 3).	 Selective	
hydrolysis	 of	 methyl	 ester	 group	 in	 compound	 3	 by	 lithium	
hydroxide	 in	methonolic	water	to	get	8‐(tert‐butoxycarbonyl)‐
3‐methyl‐2‐oxo‐1‐oxa‐8‐azaspiro[4.5]dec‐3‐ene‐4‐carboxylic	
acid	(4)	in	89%	yield.	Coupling	of	compound	4	with	6‐fluoro‐3‐
(piperidin‐4‐yl)benzo[d]isoxazole	 hydrochloride	 (5)	 in	
presence	of	EDC.HCl/HOBt	 in	dichloromethane	to	 furnish	tert‐
butyl	 4‐(4‐(6‐fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐car‐
bonyl)‐3‐methyl‐2‐oxo‐1‐oxa‐8‐azaspiro[4.5]	 dec‐3‐ene‐8‐
carboxylate	(6).	Cleavage	of	tert‐butyl	oxy	group	in	compound	
6	 by	 hydrochloric	 acid	 in	 ether	 to	 give	 4‐(4‐(6‐
fluorobenzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐3‐methyl‐
1‐oxa‐8‐azaspiro[4.5]dec‐3‐en‐2‐one	 hydrochloride	 (7).	
Acylation	 of	 compound	 7	 with	 various	 benzoyl	 chloride	
derivatives	 8	 to	 afford	 final	 products	 8‐acyl‐4‐(4‐(6‐fluoro	
benzo[d]isoxazol‐3‐yl)piperidine‐1‐carbonyl)‐3‐methyl‐1‐oxa‐
8‐azaspiro[4.5]dec‐3‐en‐2‐one	(9)	as	shown	in	Scheme	1,	Table	
1.	The	structures	of	the	synthesized	compounds	are	established	
with	the	help	of	spectral	data.	

	

	
Figure	5.	Inhibition	of	PLA2	induced	haemolysis.	

	
Compounds	 9a‐b	 and	 9d‐g	 showed	 good	 antibacterial	

activity	 against	 Escherichia	 coli,	 Klebsiella	 pneumonia,	
Salmonella	typhi,	Shigella	flexneri	and	Bacillus	subtilis.	While	the	
compounds	 9c,	 9h,	 9i	 and	 9j	 showed	moderate	 antibacterial	
activity.	 Compound	 with	 dinitro	 substituent	 showed	 highest	
antibacterial	 activity.	 Most	 of	 the	 compounds	 exhibited	
antibacterial	activity	probably	due	to	the	presence	of	bioactive	
benzisoxazole	moiety.	In	all	the	anti‐oxidant	assays	compounds	
9b,	 9f	 and	 9h	 containing	 electron	 donating	 groups	 exhibited	
good	 inhibitory	 activity.	 The	 remaining	 compounds	 showed	
moderate	anti‐oxidant	activity.	At	this	stage,	it	is	not	possible	to	
give	 any	 rational	 explanation	 for	 the	 anti‐oxidant	 activities	 of	
benzisoxazole	 derivatives	 even	 in	 the	 absence	 of	 essential	
phenolic	 group.	 In	 both	 lipoxygenase	 inhibition	 and	
phospholipase	 A2	 inhibition	 assays	 compounds	 bearing	
electron	 withdrawing	 groups	 9e	 and	9g	 exhibited	 good	 anti‐
inflammatory	 activity,	 the	 remaining	 compounds	 showed	
moderate	activity	probably	due	 to	 the	absence	of	deactivating	
groups	 on	 phenyl	 ring.	 It	 is	 interesting	 to	 note	 that	 the	



Shivaprasad	et	al.	/	European	Journal	of	Chemistry	4	(4)	(2013)	402‐407	 407	
 

compounds	 bearing	 activating	 groups	 on	 phenyl	 ring	 showed	
good	 anti‐oxidant	 activity,	 whereas	 those	 with	 deactivating	
groups	exhibited	anti‐inflammatory	activities.	
	
4.	Conclusion	
	

In	 summary,	 we	 have	 synthesized	 a	 series	 of	 new	
benzisoxazole	derivatives	in	good	yields	and	screened	them	for	
antibacterial,	 antioxidant	 and	 anti‐inflammatory	 activity.	
Compounds	9a‐b	 and	9d‐g	showed	good	antibacterial	 activity	
against	Escherichia	coli,	Klebsiella	pneumonia,	Salmonella	typhi,	
Shigella	flexneri	and	Bacillus	subtilis.	Benzisoxazoles	9b,	9f	and	
9h	 bearing	 electron	 donating	 groups	 exhibited	 prominent	
antioxidant	 activity	 and	 9e	 and	 9g	 showed	 good	 anti‐
inflammatory	activity.	
	
Acknowledgements	
	

Authors	 are	 grateful	 to	 Board	 of	 Research	 in	 Nuclear	
Sciences	 (BRNS),	 University	 Grants	 Commission	 (UGC)	 and	
Indo‐French	 Centre	 for	 the	 Promotion	 of	 Advance	 Research	
(IFCPAR),	 Government	 of	 India	 for	 financial	 support	 to	
Kanchugarakoppal	 Subbegowda	 Rangappa	 under	 the	 projects	
vide	 No.	 2009/37/40/BRNS/2266	 dated	 23‐11‐2009,	 F‐39‐
106/2010	 (SR)	 dated	 24‐12‐2010	 and	 No.	 IFC/4303‐1/2010‐
11	 Dated	 22‐12‐2010.	 Chakrabhavi	 Dhananjaya	 Mohan	 thank	
Department	 of	 Science	 and	 Technology	 for	 Innovation	 in	
Science	 Pursuit	 for	 Inspired	 Research	 fellowship	 and	
Toreshettahally	 Ramesh	 Swaroop	 thank	 Council	 of	 Scientific	
and	 Industrial	 Research	 for	 Junior	 and	 Senior	 Research	
Fellowship.	
	
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